Antibodies directed against ICOS and uses thereof

ABSTRACT

The present invention provides antibodies directed against ICOS or a derivative thereof which neutralize ICOS engagement on Treg by inhibiting the fixation between ICOS and ICOS-L and abrogate proliferation of Treg induced by plasmacytoid dendritic cells. The present invention further provides antibodies directed against ICOS or a derivative thereof which induce IL-10 and IFNγ production, induce CD4+ T cells proliferation, reduce Tconv proliferation, and increase the immunosuppressive function of Treg.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/165,152 filed May 26, 2016, now U.S. Pat. No.9,676,852, which was a divisional application of U.S. patent applicationSer. No. 14/008,423 filed Dec. 10, 2013, now U.S. Pat. No. 9,376,493,which was a Rule 371 national stage filing of PCT/EP2012/055735 filedMar. 29, 2012.

FIELD OF THE INVENTION

The invention relates to antibodies directed against ICOS and usesthereof.

BACKGROUND OF THE INVENTION

In several cancers, the establishment of an immunosuppressive T cellresponse is correlated with a poor prognosis and disease progression.

Among the different cellular effectors involved in the establishment ofimmune tolerance, the CD4⁺ regulatory T lymphocytes subset (Treg) isspecialised in the suppression of the other T cell (Tconv) as well asdendritic function. Said suppression may be correlated with a poorsurvival rate of patient suffering from cancer, especially from breastcancer.

It has been shown that large amounts of IL-10 and low quantities of IFNγproduced by CD4⁺ T cells are associated with reduced CD8⁺ T cellcytotoxic capacity, lower T cells proliferation and participate tomonocytes differentiation into immunosuppressive M2c type macrophages,related to the Tumor Associated Macrophage (TAM).

The inventors previously reported that memory CD3⁺ CD4⁺ T cells thatencompass large amounts of Treg (Ta-Treg) infiltrated primary breasttumors. Primary breast tumor infiltration by Ta-Treg and plasmacytoid DC(pDC) are both associated with poor prognosis and poor survival of thepatient suffering from breast tumors.

The inventors further confirmed that immunosuppressive mechanismsinvolving Treg are observed in most cancers and chronic infections.These suppressive mechanisms prevent an efficient immune responseagainst cancer and chronic viral infection.

Currently Treg are targeted in cancers and chronic infections using celltherapy, anti-CD25 mAbs or low doses chemotherapy. However, saidstrategies did not provide acceptable results.

In addition, it has been reported that Treg might have an important rolein diseases associated with or caused by an excessive immune response.

However, there is currently no available and efficient strategy fortreating Treg associated diseases. There is still thus a great need forproviding efficient therapeutic strategies targeting diseases involvingTreg.

SUMMARY OF THE INVENTION

Surprisingly, the inventors have shown that the interaction between ICOSand its ligand plays a central role in the activation, proliferation andsuppressive function of Treg in some cancers through interaction withplasmacytoid dendritic cells (pDC). They then concentrated their effortto generate specific antibodies with antagonist and agonist effects.

The antagonist antibodies are efficient for treating a disease or acondition associated with Treg mediated suppression of immune response.The agonist antibodies are efficient for treating a disease or acondition associated with or caused by an excessive immune response.

The present invention thus relates to an antibody directed against ICOSor a derivative thereof which:

-   -   neutralizes ICOS engagement on Treg by inhibiting the fixation        between ICOS and ICOS-L; and    -   abrogates proliferation of Treg induced by pDC.        In the context of the present invention, said antibody may also        be called “antagonist antibody”.

The invention further relates to an antibody directed against ICOS,wherein said antibody is selected from the group consisting of Icos145-1 and Icos 314-8, respectively obtainable from the hybridomadeposited at the “Collection Nationale de Cultures de Microorganismes”(CNCM, Institut Pasteur, 25 rue du Docteur Roux. 75724 Paris Cedex 15,France), in accordance with the terms of Budapest Treaty, on Jul. 2,2009 under the accession numbers CNCM I-4179 and CNCM I-4180 andderivatives thereof.

The invention also relates to an antagonist antibody directed againstICOS according to the invention or a derivative thereof for use as amedicament. The invention further relates to an antagonist antibodydirected against ICOS according to the invention or a derivative thereoffor use for treating cancers or chronic infections.

The present invention further relates to an antibody directed againstICOS or a derivative thereof which:

-   -   induces IL-10 and IFNγ production;    -   induces CD4+ T cells proliferation;    -   reduces Tconv proliferation, and    -   increases the immunosuppressive function of Treg.        In the context of the present invention, said antibody may also        be called “agonist antibody”.

The invention also relates to an antibody directed against ICOS, whereinsaid antibody is selected from the group consisting of Icos 53-3, Icos88-2 and Icos 92-17, respectively obtainable from the hybridomadeposited at the “Collection Nationale de Cultures de Microorganismes”(CNCM, Institut Pasteur, 25 rue du Docteur Roux. 75724 Paris Cedex 15,France), in accordance with the terms of Budapest Treaty, on Jul. 2,2009 under the accession numbers CNCM I-4176, CNCM I-4177, CNCM I-4178and derivatives thereof.

The invention relates to an agonist antibody according to the inventionor a derivative thereof for use as a medicament. The invention alsorelates to an agonist antibody according to the invention or aderivative thereof for use for treating autoimmune diseases,transplantation rejection or a graft versus host disease.

DETAILED DESCRIPTION OF THE INVENTION Definition

As used herein, the terms “ICOS” or “Inductible T cell costimulator”refer to a transmembrane homodimeric glycoprotein of 55 to 60 kDa whichpresents an IgV type domain in its extracellular part and a tyrosinewithin an YMFM motif in its cytoplasmic part. It has been shown thatICOS engagement with its ligand induces the phosphorylation of thetyrosine in the cytoplasmic part of ICOS. Said phosphorylation isresponsible for the recruitment of the p85 PI3K regulatory subunit,which activates the PI3K/AKT signaling pathway.

ICOS engagement is also described to induce the expression of CD40L atthe cell surface. CD40L is known to have an important effect in thecooperation between T lymphocytes and B lymphocytes.

ICOS has been found to be expressed, following TCR activation, onconventional T cells (Tconv CD4+, CD8+ subsets) as well as on Treg. Theinventors showed that said activation was more important in patientssuffering from melanoma or breast cancer.

As used herein, the terms “ICOSL”. “ICOS-L” and “B7-H2” refer to an ICOSligand. Said ligand is present on lymphoid cells such as B lymphocytes,macrophages, dendritic cells, as well as on non-lymphoid cells such asendothelial or epithelial cells. ICOS engagement has an important rolein the lymphocyte activation, and it induces the proliferation andsurvival of T lymphocytes, especially Treg.

As used herein, the term “JICOS 1” refers to a specific cell lineexpressing ICOS.

As used herein, a “monoclonal antibody” in its various grammatical formsrefers to a population of antibodies that contains only one species ofantibody combining sites capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g. a bispecific monoclonal antibody. Although historically amonoclonal antibody was produced by immortalization of a clonally pureimmunoglobulin secreting cell line, a monoclonally pure population ofantibody molecules can also be prepared by the methods of the presentinvention. Laboratory methods for preparing monoclonal antibodies arewell known in the art (see, for example, Harlow et al., 1988).Monoclonal antibodies (mAbs) may be prepared by immunizing purifiedmutated TXAS into a mammal, e.g. a mouse, rat, human and the likemammals. The antibody-producing cells in the immunized mammal areisolated and fused with myeloma or heteromyeloma cells to produce hybridcells (hybridoma). The hybridoma cells producing the monoclonalantibodies are utilized as a source of the desired monoclonal antibody.This standard method of hybridoma culture is described in Kohler andMilstein (1975). While mAbs can be produced by hybridoma culture theinvention is not to be so limited. Also contemplated is the use of mAbsproduced by an expressing nucleic acid cloned from a hybridoma of thisinvention. That is, the nucleic acid expressing the molecules secretedby a hybridoma of this invention can be transferred into another cellline to produce a transformant. The transformant is genotypicallydistinct from the original hybridoma but is also capable of producingantibody molecules of this invention, including immunologically activefragments of whole antibody molecules, corresponding to those secretedby the hybridoma. See, for example, U.S. Pat. No. 4,642,334 to Reading;PCT Publication No.; European Patent Publications No. 0239400 to Winteret al. and No. 0125023 to Cabilly et al. Antibody generation techniquesnot involving immunisation are also contemplated such as for exampleusing phage display technology to examine naive libraries (fromnon-immunised animals); see Barbas et al. (1992), and Waterhouse et al.(1993).

As used herein, the expression “anti-ICOS antibody” refers to amonoclonal antibody directed against ICOS, preferably obtained usingrecombinant ICOS-Fc as immunogen.

As used herein, the expression “derivative of an antibody” refers to anantibody which comprises the 6 CDRs of said antibody.

As used herein, the expression “53.3 mAb” or “Icos 53-3” refers to amonoclonal antibody directed against ICOS deposited at the CNCM on Jul.2, 2009 under the accession number CNCN I-4176. Said antibody is anagonist of ICOS. The expression “a derivative of 53.3 mAb” refers to ananti-ICOS antibody which comprises the 6 CDRs of 53.3 mAb.

As used herein, the expression “88.2 mAb” or “Icos 88-2” refers to amonoclonal antibody directed against ICOS deposited at the CNCM on Jul.2, 2009 under the accession number CNCN I-4177. Said antibody is anagonist of ICOS. The inventors have shown that use of said antibody inpresence of IL-2 favors Treg proliferation and the IL-10 secretion. Theexpression “a derivative of 88.2 mAb” refers to an anti-ICOS antibodywhich comprises the 6 CDRs of 88.2 mAb.

The 6 CDRs of 88.2 mAb are as in Table 1 below:

TABLE 1 Aminoacid DNA sequence sequence H-CDR1GGCTACAGTTTCACCAGCTACTGGATA GYSFTSYWIN AAC (SEQ ID NO: 17)(SEQ ID NO: 23) H-CDR2 AATATTTATCCTTCTGATACTTATACT NIYPSDSYTNYNQMFAACTACAATCAAATGTICAAGGAC KD (SEQ ID NO: 18) (SEQ ID NO: 24) H-CDR3TGGAATCTTTCTTATTACTTCGATAAT WNLSYYFDNNYYLDY AACTACTACTTGGACTAC(SEQ ID NO: 25) (SEQ ID NO: 19) L-CDR1 AGGTCTAGTAAGAGTCTCCTGCATAGTRSSKSLLHSNGNTYL AATGGCAACACTTACTTGTAT Y (SEQ ID NO: 20) (SEQ ID NO: 26)L-CDR2 CGGATGTCCAACCTTGCCTCA RMSNLAS (SEQ ID NO: 21) (SEQ ID NO: 27)L-CDR3 ATGCAACATCTAGAATATCCCTGGACG MQHLEYPWT (SEQ ID NO: 22)(SEQ ID NO: 28)

As used herein, the expression “92.17 mAb” or “Icos 92-17” refers to amonoclonal antibody directed against ICOS deposited at the CNCM on Jul.2, 2009 under the accession number CNCN I-4178. Said antibody is anagonist of ICOS. The expression “a derivative of 92.17 mAb” refers to ananti-ICOS antibody which comprises the 6 CDRs of 92.17 mAb.

As used herein, the expression “145.1 mAb” or “Icos 145-1” refers to amonoclonal antibody directed against ICOS deposited at the CNCM on Jul.2, 2009 under the accession number CNCN I-4179. Said antibody is anantagonist of ICOS. The expression “a derivative of 145.1 mAb” refers toan anti-ICOS antibody which comprises the 6 CDRs of 145-1 mAb.

As used herein, the expression “314.8 mAb” or “Icos 314-8” refer to amonoclonal antibody directed against ICOS deposited to CNCM on Jul. 2,2009 under the accession number CNCM I-4180. The inventors have shownthat use of said antibody blocks the secretion of IL-10 by Tconv. Saidantibody is an antagonist of ICOS and is highly adapted for preventingdendritic cells mediated regulatory T cells expansion and suppressivefunction in cancer such as breast cancer. The expression “a derivativeof 314.8 mAb” refers to an anti-ICOS antibody which comprises the 6 CDRsof 314.8 mAb.

The 6 CDRs of 314.8 mAb are as in Table 2 below:

TABLE 2 Aminoacid DNA sequence sequence H-CDR1GGCTACACCTTCACCACCTACTGGAT GYTFTTYWMH GCAC (SEQ ID NO: 7) (SEQ ID NO: 1)H-CDR2 GAGATTGATCCTTCTGATAGTTATGT EIDPSDSYVNYNQNTAACTACAATCAAAACTTTAAGGGC FKG (SEQ ID NO: 2) (SEQ ID NO: 8) H-CDR3TTTGATTAC FDY (SEQ ID NO: 3) (SEQ ID NO: 9) L-CDR1AGGTCTAGTAAGAGTCCCCTGCATAG ESSKSPLHSNGNIY TAACGGCAACATTTACTTATAT LY(SEQ ID NO: 4) (SEQ ID NO: 10) L-CDR2 CGGATGTCCAACCTTGCCTCA RMSNLAS(SEQ ID NO: 5) (SEQ ID NO: 11) L-CDR3 ATGCAACATCTAGAATATCCGTACACGMQHLEYPYT (SEQ ID NO: 6) (SEQ ID NO: 12)

As used herein, the expression “an antibody of the invention” refers to:

-   -   an antibody directed against ICOS able to neutralize ICOS        engagement on Treg by inhibiting the fixation between ICOS and        ICOS-L and to abrogate proliferation of Treg induced by        plasmacytoid dendritic cells, i.e. an antagonist antibody; as        well as    -   an antibody directed against ICOS able to induce IL-10 and IFNγ        production, to induce CD4⁺ T cells proliferation; to reduce        Tconv proliferation, and to increase the immunosuppressive        function of Treg, i.e. an agonist antibody.        Said expression also encompasses any derivatives of said        antibodies.

Preferably, the antibodies of the invention are chosen from 53.3 mAb,88.2 mAb, 92.17 mAb, 145.1 mAb, 145.1 mAb and 314.8 mAb and thederivatives thereof.

As used herein, the expression “antagonist antibody directed againstICOS” refers to an antibody which is able to hind to ICOS withouttriggering a cellular response similar to the response induced by thenaturally occurring ICOS. The expression “the antagonist antibodies ofthe invention” refers to 145.1 mAb, 314.8 mAb and derivatives thereof.

As used herein, the expression “agonist antibody directed against ICOS”refers to an antibody which is able to bind to ICOS and to trigger acellular response similar to the response induced by the naturallyoccurring ICOS. Said antibody thus mimics the action of ICOS. Theexpression “the agonist antibody of the invention” refers to 53.3 mAb,88.2 mAb, 92.17 mAb and derivatives thereof.

As used herein, the expressions “antigen presenting cell” and “APC”refer to a class of immune cells capable of internalizing and processingan antigen, so that antigenic determinants are presented on the surfaceof the cell as MHC-associated complexes, in a manner capable of beingrecognized by the immune system (e. g., MHC class I restricted cytotoxicT lymphocytes and/or MHC class II restricted helper T lymphocytes). Thetwo requisite properties that allow a cell to function as an APC are theability to process endocytosed antigens and the expression of MHC geneproducts. Examples of APC include dendritic cells (DC), mononuclearphagocytes (e. g. macrophages), B lymphocytes, Langerhans cells of theskin and, in humans, endothelial cells.

As used herein, the expressions “Treg” and “Regulatory T cells” refer toa specific population of T lymphocytes that have the capacity todominantly suppress the proliferation of responder T cells in vitro andinhibit autoimmune diseases. Treg have been implicated as majorcontributors to the ultimate failure of anti-tumor immune responses inhumans. For instance, in ovarian cancer, Treg suppress tumor-specific Tcells and high numbers of tumor-associated Treg are associated withreduced survival time. The inventors have shown that Treg selectivelyinhibit the host immune response and thereby contribute to cancerprogression, especially in breast cancer. Treg were originallyidentified as a CD4⁺ CD25⁺ cell population, but are also characterizedby the expression of the forkhead family transcription factor, FoxP3.

The inventors have shown that Treg proliferate in situ within cancertissue of a patient and express the cell surface markers ICOS and CD39,compared to Treg extracted from blood of the same patient.

By opposition, the term “Tconv” refers to T cells other than Treg. Theterm “Tconv” thus includes T cells which function to eliminate antigen(e.g. by producing cytokines which modulate the activation of othercells or by cytotoxic activity). This term includes Thelper cells (e.g.ThI and Th2 cells) and cytotoxic T cells. In this respect, Thelper cellspreferably express CD4 and express low or undetectable levels of CD25.CTL cells preferably express CD8 and low or undetectable levels of CD4.Preferably, a non-Treg cell does not express both CD4 and CD25.Preferably, a non-Treg cell does not express FoxP3.

As used herein, the expressions “tumor associated regulatory T cells”and “Ta-Treg” refer to Regulatory T cells associated with tumors, forexample with breast tumors. The inventors have indeed shown that Ta-Tregare present in the lymphoid infiltrates of mammary tumoral tissue andpresent a negative impact in the survival of the patient suffering frombreast cancer.

As used herein, the expressions “plasmacytoid dendritic cells” and “pDC”refer to innate immune cells that circulate in the blood and are foundin peripheral lymphoid organs. They constitute a group of cellsbelonging to the peripheral blood mononuclear cells (PBMC) group.

As used herein, the expressions “Tumor associated plasmacytoid dendriticcells” and “Ta-pDC” refer to plasmacytoid dendridic cells associatedwith tumors, for example mammary tumors. The inventors have shown thatTa-pDC are able to induce the proliferation of Ta-Treg under thedependence of the ICOS/ICOSL co-stimulation.

As used herein, the terms “IL-10” and “interleukin-10” refer to a humancytokine synthesis inhibitory factor (CSIF), which is ananti-inflammatory cytokine. This cytokine is primarily produced bymonocytes and to a lesser extent by lymphocytes. This cytokine haspleiotropic effects in immunoregulation and inflammation. Itdown-regulates the expression of Th1 cytokines, MHC class II antigens.It also enhances B cell survival, proliferation, and antibodyproduction. This cytokine can block NF-κB activity, and is involved inthe regulation of the JAK-STAT signaling pathway.

As used herein, the terms “IFNγ” and “interferon-gamma” refer to adimeric protein with subunits of 146 amino acids. The importance ofIFN-γ in the immune system stems in part from its ability to inhibitviral replication directly, and most importantly from itsimmunostimulatory and immunomodulatory effects. IFNγ is producedpredominantly by natural killer (NK) and natural killer T (NKT) cells aspart of the innate immune response, and by CD4 and CD8 cytotoxic Tlymphocyte (CTL) effector T cells once antigen-specific immunitydevelops.

As used herein, the terms “treating” or “treatment” means reversing,alleviating, inhibiting the progress of, or preventing the disorder orcondition to which such term applies, or one or more symptoms of suchdisorder or condition.

A “therapeutically effective amount” is intended for a minimal amount ofactive agent which is necessary to impart therapeutic benefit to asubject. For example, a “therapeutically effective amount” is an amountwhich induces, ameliorates or otherwise causes an improvement in thepathological symptoms, disease progression or physiological conditionsassociated with a disease or which improves resistance to a disorder.

As used herein, the term “prevention” refers to alleviating the diseaseor condition from occurring in a subject which has not yet beendiagnosed as having it. As used herein, the term “subject” denotes amammal, such as a rodent, a feline, a canine, and a primate. Preferablya subject according to the invention is a human.

The term “cancer” includes malignancies of the various organ systems,such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, andgenito-urinary tract, as well as adenocarcinomas which includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer and/or testicular tumors, non-small cell carcinoma of the lung,cancer of the small intestine and cancer of the esophagus.

The term “Treg associated disease” shall be taken to encompass anydisease or disorder or state in which modulation of Treg numbers and/oractivity may provide a beneficial effect. This term encompasses:

-   -   diseases and conditions associated with Treg mediated        suppression of a subject's immune response,    -   diseases and conditions associated with or caused by an        excessive immune response.

As used herein, the expression “diseases and conditions associated withTreg mediated suppression of immune response” are diseases andconditions caused by the Treg suppression of the proliferation ofimmunomodulating cells such as tumor-specific T cell. As previouslymentioned, the inventors have shown that Treg are associated with a poordiagnostic and survival rate in a patient suffering from cancer.

Non limiting examples of diseases and conditions associated with Tregmediated suppression of a subject's immune system are cancer and chronicinfections.

As used herein, “diseases and conditions associated with or caused by anexcessive immune response” are for example autoimmune diseases,transplantation rejection or a graft versus host disease.

This expression further encompasses inflammatory conditions, such asinflammatory disorder of the nervous system (e.g. multiple sclerosis),mucosal inflammatory disease (e.g. inflammatory bowel disease, asthma ortonsillitis), inflammatory skin disease (e.g. dermatitis, psoriasis orcontact hypersensitivity), autoimmune arthritis (e.g. rheumatoidarthritis).

As used herein, the term “immune response” refers to the concertedaction of lymphocytes, antigen presenting cells, phagocytic cells,granulocytes, and soluble macromolecules produced by the above cells orthe liver (including antibodies, cytokines, and complement) that resultsin selective damage to, destruction of, or elimination from the body ofa subject of cancerous cells, metastatic tumor cells, malignantmelanoma, invading pathogens, cells or tissues infected with pathogens,or, in cases of autoimmunity or pathological inflammation, normal cellsor tissues of a subject.

As used herein, an “autoimmune disease” is a disease or a disorderarising from and directed against an individual's own tissues.

Antagonist Antibodies of the Invention

It has been shown that ICOS-L, which is a specific ligand of ICOS, isexpressed on plasmacytoid dendritic cells. The inventors have shown thatTumor associated Treg were in close contact with Tumor-associatedplasmacytoid dendritic cells, indicating that such an interaction allowsthe engagement of ICOS with ICOS-L in tumors. They further showed thatin situ ICOS/ICOS-L interaction leads to ICOS-L downregulation on theTa-pDC membrane. The inventors have developed an antagonist antibodydirected against ICOS and showed that the addition of said antibodyabrogates totally the ICOS-L downregulation on pDC, which is responsiblefor Ta-Treg activation and proliferation.

The inventors have shown that the antagonist antibody according to theinvention neutralizes ICOS engagement on Treg and abrogates theirexpansion induced by pDC. More precisely, said antibody abrogates Tregproliferation and IL-10 secretion induced by ICOS/ICOSL interaction.

The antagonist antibodies of the invention are thus highly appropriatefor abrogating the immunosuppressive response involved in pathologicalmechanism. They are thus useful for treating diseases and conditionsassociated with Treg mediated suppression of immune response.

The invention is thus drawn to an antibody directed against ICOS andderivatives thereof which:

-   -   neutralizes ICOS engagement on Treg by inhibiting the fixation        between ICOS and ICOS-L; and    -   abrogates proliferation of Treg induced by plasmacytoid        dendritic cell.

In an embodiment, said antibody is a monoclonal antibody.

In an embodiment, said antibody is a chimeric antibody.

In an embodiment, said antibody is a humanized antibody.

By “neutralizing ICOS engagement on Treg”, it is meant that the antibodyinterferes with the cooperation between ICOS and its ligand ICOS-L.

By “abrogating proliferation of Treg”, it is meant that a significantdecrease, preferably a total stop, of the proliferation of Treg isobserved in a target tissue, preferably a tumor tissue, as compared to acontrol tissue, preferably a non-tumor tissue, more preferably blood.

The invention further relates to an antibody directed against ICOS,wherein said antibody is selected from the group consisting of Icos145-1 and Icos 314-8, respectively obtainable from the hybridomadeposited at the CNCM on Jul. 2, 2009 under the accession numbers CNCMI-4179 and CNCM I-4180 and derivatives thereof.

The invention also relates to an antibody which comprises the 6 CDRs ofan antibody selected from the group consisting of Icos 145-1 and Icos314-8, respectively obtainable from the hybridoma deposited at the CNCMon Jul. 2, 2009 under the accession numbers CNCM I-4179 and CNCM I-4180and derivatives thereof.

The invention also relates to an antibody which comprises the 6 CDRs ofTable 2 above.

In another embodiment, the invention relates to a derivative antibody ofone of the antibodies selected from the group consisting of Icos 145-1and Icos 314-8, respectively obtainable from the hybridoma deposited atthe CNCM on Jul. 2, 2009 under the accession numbers CNCM I-4179 andCNCM I-4180.

Therapeutic Use of Antagonist Antibodies of the Invention

By neutralizing ICOS engagement on Treg and abrogating proliferation ofTreg, the antagonist antibodies of the invention are highly appropriatefor use for treating diseases and conditions associated with Tregmediated suppression of immune response, for example cancers and chronicinfections. Said antibodies may thus be used for restoring an anti-tumorimmunity.

The invention therefore relates to the antagonist antibody directedagainst ICOS according to the invention or a derivative thereof for useas a medicament.

The invention further relates to the antagonist antibody directedagainst ICOS according to the invention or a derivative thereof for usefor treating disease or a condition associated with Treg mediatedsuppression of immune response.

In a preferred embodiment, said disease or a condition associated withTreg mediated suppression of immune response is a disease selected inthe group consisting of cancers and chronic infections.

Indeed, the inventors have shown that the antagonist antibodies of theinvention are adapted for modulating Treg numbers and/or activity sothat to abrogate the immunosuppressive effect related to those Treg.Therefore, said antagonist antibodies represent a highly promisingstrategy for treating diseases associated with a suppression of immunesystem such as cancer and chronic infections.

Examples of cancers include, but are not limited to human malignantlymphoma, breast cancer, ovarian cancer, colon cancer lung cancer, braincancer, prostate cancer, head and neck cancer, pancreatic cancer,bladder cancer, colorectal cancer, bone cancer, cervical cancer, livercancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer,stomach cancer, testicular cancer and skin cancer.

Examples of chronic infections include, but are not limited to, viral,bacterial, parasitic or fungal infections such as chronic hepatitis,lung infections, lower respiratory tract infections, bronchitis,influenza, pneumoniae and sexually transmitted diseases. Examples ofviral infections include, but are not limited to, hepatitis (HAV, HBV,HCV), herpes simplex (HSV), herpes zoster, HPV, influenza (Flu), AIDSand AIDS related complex, chickenpox (varicella), common cold,cytomegalovirus (CMV) infection, smallpox (variola), Colorado tickfever, dengue fever, ebola hemorrhagic fever, foot and mouth disease,lassa fever, measles, marburg hemorrhagic fever, infectiousmononucleosis, mumps, norovirus, poliomyelitis, progressive multifocalleukencephalopathy (PML), rabies, rubella, SARS, viral encephalitis,viral gastroenteritis, viral meningitis, viral pneumonia, West Niledisease and yellow fever.

Examples of bacterial infections include, but are not limited to,pneumonia, bacterial meningitis, cholera, diphtheria, tuberculosis,anthrax, botulism, brucellosis, campylobacteriosis, typhus, gonorrhea,listeriosis, Lyme disease, rheumatic fever, pertussis (Whooping Cough),plague, salmonellosis, scarlet fever, shigellosis, syphilis, tetanus,trachoma, tularemia, typhoid fever, and urinary tract infections.

Examples of parasitic infections include, but are not limited to,malaria, leishmaniasis, trypanosomiasis, chagas disease,cryptosporidiosis, fascioliasis, filariasis, amebic infections,giardiasis, pinworm infection, schistosomiasis, taeniasis,toxoplasmosis, trichinellosis, and trypanosomiasis. Examples of fungalinfections include, but are not limited to, candidiasis, aspergillosis,coccidioidomycosis, cryptococcosis, histoplasmosis and tinea pedis.

In a preferred embodiment of the invention, the invention relates to theantagonist antibodies directed against ICOS according to the inventionor a derivative thereof for use for treating cancer. Preferably, saidcancer is selected from human malignant lymphoma, ovarian cancer,cervical cancer and breast cancer. Most preferably, said cancer isbreast cancer.

The invention also relates to a method for treating disease or acondition associated with Treg mediated suppression of immune responseis a disease selected in the group consisting of cancers and chronicinfections, preferably cancers and chronic infections, preferablycancers, wherein said method comprises the step of administering to asubject in need thereof a therapeutically effective amount of anantagonist antibody directed against ICOS according to the invention ora derivative thereof.

Agonist Antibodies Directed Against ICOS

ICOS engagement has been found to be associated with animmunosuppressive T cell response. Indeed, said engagement has beendescribed to reduce IL-10 and IFNγ production and to reduce CD4⁺ T cellproliferation.

Therefore, as evidenced by the inventors, an agonist antibody of ICOSprovides the opposite effect and is beneficial for treating diseasesassociated with or caused by an excessive immune response. The inventionthus relates to an antibody directed against ICOS or a derivativethereof which:

-   -   induces IL-10 and IFNγ production;    -   induces CD4+ T cell proliferation;    -   reduces Tconv proliferation, and    -   increases the immunosuppressive function of Treg.

By “inducing IL-10 and IFNγ production”, it is meant that a significantincrease of the production of IL-10 and IFNγ is observed.

By “inducing CD4+ T cell proliferation”, it is meant that a significantincrease of the proliferation of CD4+ T cells is observed in a targettissue, preferably a tumor tissue, as compared to a control tissue,preferably a non-tumor tissue, more preferably blood.

By “reducing Tconv proliferation”, it is meant that a significantdecrease of the proliferation of Tconv is observed in a target tissue,preferably a tumor tissue, as compared to a control tissue, preferably anon-tumor tissue, more preferably blood.

By “increasing the immunosuppressive function of Treg”, it is meant thata significant increase of the Treg suppressive activity is observed.

In an embodiment, said antibody is a monoclonal antibody.

In an embodiment, said antibody is a chimeric antibody.

In an embodiment, said antibody is a humanized antibody.

The invention further relates to an antibody directed against ICOS,wherein said antibody is selected from the group consisting of Icos53-3, Icos 88-2 and Icos 92-17, respectively obtainable from thehybridoma deposited at the CNCM on Jul. 2, 2009 under the accessionnumbers CNCM I-4176, CNCM I-4177, CNCM I-4178 and derivatives thereof.

The invention also relates to an antibody which comprises the 6 CDRs ofan antibody selected from the group consisting of Icos 53-3, Icos 88-2and Icos 92-17 respectively obtainable from the hybridoma deposited atthe CNCM on Jul. 2, 2009 under the accession numbers CNCM I-4176, CNCMI-4177, CNCM I-4178.

The invention also relates to an antibody which comprises the 6 CDRs ofTable 1 above.

In another embodiment, the invention relates to a derivative antibody ofone of the antibodies selected from the group consisting of Icos 53-3,Icos 88-2 and Icos 92-17, respectively obtainable from the hybridomadeposited at the CNCM on Jul. 2, 2009 under the accession numbers CNCMI-4176, CNCM I-4177, CNCM I-4178.

Therapeutic Use of Agonist Antibodies of the Invention

The invention also relates to the agonist antibody directed against ICOSaccording to the invention or a derivative thereof for use as amedicament.

The invention is also drawn to the agonist antibody directed againstICOS according to the invention or a derivative thereof for use fortreating a disease or a condition associated with or caused by anexcessive immune response.

The invention is also drawn to the agonist antibody directed againstICOS according to the invention or a derivative thereof for use fortreating an autoimmune disease, transplantation rejection or a graftversus host disease.

In one particular embodiment, said autoimmune disease is selected fromthe group consisting of rheumatoid arthritis (RA), insulin dependentdiabetes mellitus (Type 1 diabetes), multiple sclerosis (MS), Crohn'sdisease, systemic lupus erythematosus (SLE), scleroderma, Sjogren'ssyndrome, pemphigus vulgaris, pemphigoid, addison's disease, ankylosingspondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmunehepatitis, coeliac disease, dermatomyositis, Goodpasture's syndrome,Graves' disease, Guillain-Barre syndrome, Hashimoto's disease,idiopathic leucopenia, idiopathic thrombocytopenic purpura, maleinfertility, mixed connective tissue disease, myasthenia gravis,pernicious anemia, phacogenic uveitis, primary biliary cirrhosis,primary myxoedema, Reiter's syndrome, stiff man syndrome,thyrotoxicosis, ulceritive colitis, and Wegener's granulomatosis.

In another embodiment, the invention is also drawn to the agonistantibody directed against ICOS according to the invention or aderivative thereof for use for treating an inflammatory disorderselected in the group consisting of inflammatory disorder of the nervoussystem such as multiple sclerosis, mucosal inflammatory disease such asinflammatory bowel disease, asthma or tonsillitis, inflammatory skindisease such as dermatitis, psoriasis or contact hypersensitivity, andautoimmune arthritis such as rheumatoid arthritis.

The invention also relates to a method for treating a disease or acondition associated with or caused by an excessive immune response,preferably an autoimmune disease, a transplantation rejection, a graftversus host disease, or a inflammatory disorder wherein said methodcomprises the step of administering to a subject in need thereof atherapeutically effective amount of an agonist antibody directed againstICOS according to the invention or a derivative thereof.

Nucleic Acid Sequence Encoding an Antibody of the Invention

A further embodiment of the invention relates to a nucleic acid sequenceencoding an antibody one of the antibodies selected from the groupconsisting of 53.3 mAb, 88.2 mAb, 92.17 mAb, 145.1 mAb, 314.8 mAb andderivatives thereof.

In a particular embodiment, the invention relates to a nucleic acidsequence encoding the VH domain or the VL domain of one of theantibodies selected from the group consisting of 53.3 mAb, 88.2 mAb,92.17 mAb, 145.1 mAb, 314.8 mAb and derivatives thereof.

Typically, said nucleic acid is a DNA or RNA molecule, which may beincluded in any suitable vector, such as a plasmid, cosmid, episome,artificial chromosome, phage or a viral vector.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. So, a further object of the invention relates to a vectorcomprising a nucleic acid of the invention. Such vectors may compriseregulatory elements, such as a promoter, enhancer, terminator and thelike, to cause or direct expression of said antibody upon administrationto a subject. Examples of promoters and enhancers used in the expressionvector for animal cell include early promoter and enhancer of SV40, LTRpromoter and enhancer of Moloney mouse leukemia virus, promoter andenhancer of immunoglobulin H chain and the like.

Any expression vector for animal cell can be used, so long as a geneencoding the human antibody C region can be inserted and expressed.Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR,pSG1 beta d2-4- and the like. Other examples of plasmids includereplicating plasmids comprising an origin of replication, or integrativeplasmids, such as for instance pUC, pcDNA, pBR, and the like. Otherexamples of viral vector include adenoviral, retroviral, herpes virusand AAV vectors. Such recombinant viruses may be produced by techniquesknown in the art, such as by transfecting packaging cells or bytransient transfection with helper plasmids or viruses. Typical examplesof virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+cells, 293 cells, etc. Detailed protocols for producing suchreplication-defective recombinant viruses may be found for instance inWO 95/14785, WO 96/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No.6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94/19478.

A further object of the present invention relates to a cell which hasbeen transfected, infected or transformed by a nucleic acid and/or avector according to the invention. The term “transformation” means theintroduction of a “foreign” (i.e. extrinsic or extracellular) gene, DNAor RNA sequence to a host cell, so that the host cell will express theintroduced gene or sequence to produce a desired substance, typically aprotein or enzyme coded by the introduced gene or sequence. A host cellthat receives and expresses introduced DNA or RNA has been“transformed”. The nucleic acids of the invention may be used to producean antibody of the invention in a suitable expression system. The term“expression system” means a host cell and compatible vector undersuitable conditions, e.g. for the expression of a protein coded for byforeign DNA carried by the vector and introduced to the host cell.

Common expression systems include E. coli host cells and plasmidvectors, insect host cells and Baculovirus vectors, and mammalian hostcells and vectors. Other examples of host cells include, withoutlimitation, prokaryotic cells (such as bacteria) and eukaryotic cells(such as yeast cells, mammalian cells, insect cells, plant cells, etc.).Specific examples include E. coli, Kluyveromyces or Saccharomycesyeasts, mammalian cell lines (e.g. Vero cells, CHO cells, 3T3 cells, COScells, etc.) as well as primary or established mammalian cell cultures(e.g. produced from lymphoblasts, fibroblasts, embryonic cells,epithelial cells, nervous cells, adipocytes, etc.). Examples alsoinclude mouse SP2/0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell(ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene(hereinafter referred to as “DHFR gene”) is defective, ratYB2/3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as“YB2/0 cell”), and the like.

The present invention also relates to a method of producing arecombinant host cell expressing an antibody according to the invention,said method comprising the steps of:

(i) introducing in vitro or ex vivo a recombinant nucleic acid or avector as described above into a competent host cell,

(ii) culturing in vitro or ex vivo the recombinant host cell obtained,and

(iii) optionally, selecting the cells which express and/or secrete saidantibody. Such recombinant host cells can be used for the production ofantibodies of the invention.

Pharmaceutical Composition According to the Invention

The invention also relates to pharmaceutical compositions comprising anantibody of the invention.

Therefore, an antibody of the invention may be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form therapeuticcompositions.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for atopical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment. To prepare pharmaceutical compositions,an effective amount of the antibody may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Thepharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersions; formulations including sesame oil, peanut oilor aqueous propylene glycol; and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In allcases, the form must be sterile and must be fluid to the extent thateasy syringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

An antibody of the invention can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils.

The proper fluidity can be maintained, for example, by the use of acoating, such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization.

Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum-drying and freeze-drying techniques which yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose.

These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved in 1 mlof isotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion. (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

The antibodies of the invention may be formulated within a therapeuticmixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per doseor so. Multiple doses can also be administered. In addition to thecompounds formulated for parenteral administration, such as intravenousor intramuscular injection, other pharmaceutically acceptable formsinclude, e.g. tablets or other solids for oral administration; timerelease capsules; and any other form currently used.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the introduction of antibodies into host cells. Theformation and use of liposomes and/or nanoparticles are known to thoseof skill in the art.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) are generally designedusing polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs)). MLVs generally havediameters of from 25 nm to 4 Sonication of MLVs results in the formationof small unilamellar vesicles (SUVs) with diameters in the range of 200to 500 A, containing an aqueous solution in the core. The physicalcharacteristics of liposomes depend on pH, ionic strength and thepresence of divalent cations.

Method for Producing Antibodies of the Invention

Antibodies of the invention may be produced by any technique known inthe art, such as, without limitation, any chemical, biological, geneticor enzymatic technique, either alone or in combination.

Knowing the amino acid sequence of the desired sequence, one skilled inthe art can readily produce said antibodies, by standard techniques forproduction of polypeptides. For instance, they can be synthesized usingwell-known solid phase method, preferably using a commercially availablepeptide synthesis apparatus (such as that made by Applied Biosystems,Foster City, Calif.) and following the manufacturer's instructions.Alternatively, antibodies of the invention can be synthesized byrecombinant DNA techniques well-known in the art. For example,antibodies can be obtained as DNA expression products afterincorporation of DNA sequences encoding the antibodies into expressionvectors and introduction of such vectors into suitable eukaryotic orprokaryotic hosts that will express the desired antibodies, from whichthey can be later isolated using well-known techniques.

In particular, the invention further relates to a method of producing anantibody of the invention, which method comprises the steps consistingof:

(i) culturing a transformed host cell according to the invention underconditions suitable to allow expression of said antibody; and

(ii) recovering the expressed antibody.

In another particular embodiment, the method comprises the steps of:

(i) culturing the hybridoma deposited as CNCM I-4176, CNCM I-4177, CNCMI-4178, CNCM I-4179, or CNCM I-4180 under conditions suitable to allowexpression of the antibody; and

(ii) recovering the expressed antibody.

Antibodies of the invention are suitably separated from the culturemedium by conventional immunoglobulin purification procedures such as,for example, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

In a particular embodiment, the human chimeric antibody of the presentinvention can be produced by obtaining nucleic sequences encoding VL andVH domains as previously described, constructing a human chimericantibody expression vector by inserting them into an expression vectorfor animal cell having genes encoding human antibody CH and humanantibody CL, and expressing the coding sequence by introducing theexpression vector into an animal cell. As the CH domain of a humanchimeric antibody, it may be any region which belongs to humanimmunoglobulin, but those of IgG class are suitable and any one ofsubclasses belonging to IgG class, such as IgG1, IgG2, IgG3 and IgG4,can also be used. Also, as the CL of a human chimeric antibody, it maybe any region which belongs to Ig, and those of kappa class or lambdaclass can be used. Methods for producing chimeric antibodies involveconventional recombinant DNA and gene transfection techniques are wellknown in the art (See patent documents U.S. Pat. No. 5,202,238; and U.S.Pat. No. 5,204,244).

The humanized antibody of the present invention may be produced byobtaining nucleic acid sequences encoding CDR domains, as previouslydescribed, constructing a humanized antibody expression vector byinserting them into an expression vector for animal cell having genesencoding (i) a heavy chain constant region identical to that of a humanantibody and (ii) a light chain constant region identical to that of ahuman antibody, and expressing the genes by introducing the expressionvector into an animal cell.

The humanized antibody expression vector may be either of a type inwhich a gene encoding an antibody heavy chain and a gene encoding anantibody light chain exists on separate vectors or of a type in whichboth genes exist on the same vector (tandem type). In respect ofeasiness of construction of a humanized antibody expression vector,easiness of introduction into animal cells, and balance between theexpression levels of antibody H and L chains in animal cells, humanizedantibody expression vector of the tandem type is preferred. Examples oftandem type humanized antibody expression vector include pKANTEX93 (WO97/10354), pEE18 and the like.

Methods for producing humanized antibodies based on conventionalrecombinant DNA and gene transfection techniques are well known in theart. Antibodies can be humanized using a variety of techniques known inthe art including, for example, CDR-grafting (EP 239,400; PCTpublication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089), veneering or resurfacing (EP 592,106; EP 519,596), and chainshuffling (U.S. Pat. No. 5,565,332). The general recombinant DNAtechnology for preparation of such antibodies is also known (seeEuropean Patent Application EP 125023 and International PatentApplication WO 96/02576).

The Fab of the present invention can be obtained by treating an antibodywhich specifically reacts with ICOS with a protease, papaine. Also, theFab can be produced by inserting DNA encoding Fab of the antibody into avector for prokaryotic expression system, or for eukaryotic expressionsystem, and introducing the vector into a procaryote or eucaryote (asappropriate) to express the Fab.

The F(ab′)2 of the present invention can be obtained treating anantibody which specifically reacts with ICOS with a protease, pepsin.

Also, the F(ab′)2 can be produced by binding Fab′ described below via athioether bond or a disulfide bond.

The Fab′ of the present invention can be obtained by treating F(ab′)2which specifically reacts with human ICOS with a reducing agent,dithiothreitol. Also, the Fab′ can be produced by inserting DNA encodingFab′ fragment of the antibody into an expression vector for prokaryote,or an expression vector for eukaryote, and introducing the vector into aprokaryote or eukaryote (as appropriate) to perform its expression.

The scFv of the present invention can be produced by obtaining cDNAencoding the VH and VL domains as previously described, constructing DNAencoding scFv, inserting the DNA into an expression vector forprokaryote, or an expression vector for eukaryote, and then introducingthe expression vector into a prokaryote or eukaryote (as appropriate) toexpress the scFv. To generate a humanized scFv fragment, a well knowntechnology called CDR grafting may be used, which involves selecting thecomplementary determining regions (CDRs) from a donor scFv fragment, andgrafting them onto a human scFv fragment framework of known threedimensional structure (see, e.g., WO98/45322; WO 87/02671; U.S. Pat. No.5,859,205; U.S. Pat. No. 5,585,089; U.S. Pat. No. 4,816,567; EP0173494).

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody. Itis known that when a humanized antibody is produced by simply graftingonly CDRs in VH and VL of an antibody derived from a non-human animal inFRs of the VH and VL of a human antibody, the antigen binding activityis reduced in comparison with that of the original antibody derived froma non-human animal. It is considered that several amino acid residues ofthe VH and VL of the non-human antibody, not only in CDRs but also inFRs, are directly or indirectly associated with the antigen bindingactivity. Hence, substitution of these amino acid residues withdifferent amino acid residues derived from FRs of the VH and VL of thehuman antibody would reduce of the binding activity.

In order to resolve the problem, in antibodies grafted with human CDR,attempts have to be made to identify, among amino acid sequences of theFR of the VH and VL of human antibodies, an amino acid residue which isdirectly associated with binding to the antibody, or which interactswith an amino acid residue of CDR, or which maintains thethree-dimensional structure of the antibody and which is directlyassociated with binding to the antigen. The reduced antigen bindingactivity could be increased by replacing the identified amino acids withamino acid residues of the original antibody derived from a non-humananimal.

Modifications and changes may be made in the structure of the antibodiesof the present invention, and in the DNA sequences encoding them, andstill obtain a functional molecule that encodes an antibody withdesirable characteristics. In making the changes in the amino sequences,the hydropathic index of amino acids may be considered. The importanceof the hydropathic amino acid index in conferring interactive biologicfunction on a protein is generally understood in the art. It is acceptedthat the relative hydropathic character of the amino acid contributes tothe secondary structure of the resultant protein, which in turn definesthe interaction of the protein with other molecules, for example,enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophane (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

A further embodiment of the present invention also encompassesfunction-conservative variants of the antibodies of the presentinvention.

“Function-conservative variants” are those in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like).

Amino acids other than those indicated as conserved may differ in aprotein so that the percent protein or amino acid sequence similaritybetween any two proteins of similar function may vary and may be, forexample, from 70% to 99% as determined according to an alignment schemesuch as by the Cluster Method, wherein similarity is based on theMEGALIGN algorithm.

A “function-conservative variant” also includes a polypeptide which hasat least 60% amino acid identity as determined by BLAST or FASTAalgorithms, preferably at least 75%, more preferably at least 85%, stillpreferably at least 90%, and even more preferably at least 95%, andwhich has the same or substantially similar properties or functions asthe native or parent protein to which it is compared. Two amino acidsequences are “substantially homologous” or “substantially similar” whengreater than 80%, preferably greater than 85%, preferably greater than90% of the amino acids are identical, or greater than about 90%,preferably grater than 95%, are similar (functionally identical) overthe whole length of the shorter sequence. Preferably, the similar orhomologous sequences are identified by alignment using, for example, theGCG (Genetics Computer Group, Program Manual for the GCG Package,Version 7, Madison, Wis.) pileup program, or any of sequence comparisonalgorithms such as BLAST, FASTA, etc.

For example, certain amino acids may be substituted by other amino acidsin a protein structure without appreciable loss of activity. Since theinteractive capacity and nature of a protein define the protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and, of course, in its DNA encodingsequence, while nevertheless obtaining a protein with like properties.It is thus contemplated that various changes may be made in theantibodies sequences of the invention, or corresponding DNA sequenceswhich encode said antibodies, without appreciable loss of theirbiological activity.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. As outlined above, aminoacid substitutions are generally therefore based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like.

Exemplary substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine. Another type of amino acid modification of the antibody ofthe invention may be useful for altering the original glycosylationpattern of the antibody.

By “altering” is meant deleting one or more carbohydrate moieties foundin the antibody, and/or adding one or more glycosylation sites that arenot present in the antibody.

Glycosylation of antibodies is typically N-linked. “N-linked” refers tothe attachment of the carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serine andasparagines-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. Addition of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites). Another type of covalent modificationinvolves chemically or enzymatically coupling glycosides to theantibody. These procedures are advantageous in that they do not requireproduction of the antibody in a host cell that has glycosylationcapabilities for N- or O-linked glycosylation. Depending on the couplingmode used, the sugar(s) may be attached to (a) arginine and histidine,(b) free carboxyl groups, (c) free sulfhydryl groups such as those ofcysteine, (d) free hydroxyl groups such as those of serine, threonine,orhydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan, or (f) the amide group of glutamine. Forexample, such methods are described in WO87/05330.

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact.

Enzymatic cleavage of carbohydrate moieties on antibodies can beachieved by the use of a variety of endo- and exo-glycosidases.

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of non proteinaceous polymers, eg.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337. It may be also desirable to modifythe antibody of the invention with respect to effector function, e.g. soas to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/orcomplement dependent cytotoxicity (CDC) of the antibody. This may beachieved by introducing one or more amino acid substitutions in an Fcregion of the antibody. Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginter-chain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and/orantibody-dependent cellular cytotoxicity (ADCC) (Caron P C. et al. J ExpMed. 1992 Oct. 1; 176(4):1191-5 and Shopes B. J Immunol. 1992 May 1;148(9):2918-22).

Diagnostic Method

The present invention also relates to a diagnostic method of anincreased risk of relapse or early death in a breast cancer patient.Indeed, as shown in Example 3, the presence of high ICOS Treg cellsnumber is associated to lower Progression Free Survival or OverallSurvival for breast cancer patients.

Thus, the invention relates to a method for diagnosing an increased riskof relapse or early death in a breast cancer patient, comprising thestep of quantifying ICOS positive (ICOS⁺) Treg cells in a sample of saidpatient. If said number is high, for example greater than 1.7 ICOS⁺cells/spot when using the method of example 3 and FIG. 9, then there isan increased risk of relapse or early death in said breast cancerpatient.

The invention also relates to a method for selecting patientssusceptible of being treated by anti-ICOS immunotherapy, comprising thestep of quantifying ICOS positive Treg cells in a sample of saidpatient. Said immunotherapy may be anti-ICOS antibodies of theinvention.

Said sample may come from a biopsy. Said quantification of ICOS⁺ Tregcells may be performed thanks to anti ICOS antibodies, especially thanksto any one of the antibodies described above.

Treatment in Pre-Clinical Mammary Tumor Model

As shown in Example 6, treatment of an established murine model ofmammary tumor with a surrogate neutralizing rat anti murine ICOSantibody (17G9, IgG2b), reduces tumor progression, reinforcing thepotential of treatment with anti-ICOS neutralizing antibodies of theinvention to favor of tumor regression in the subpopulation of patientswith high ICOS⁺ Treg detection in their primary breast tumor.

The invention will further be illustrated in view of the followingfigures and example.

FIGURE LEGEND

FIG. 1: Ta-Treg strongly express ICOS, co-localized with Ta-pDC andproliferate in situ but do not proliferate in vitro.

A—Tumor frozen sections were stained with anti ICOS Ab (green) and Ki67Ab (brown) and secondary anti murine Ab conjugated to HRP and revealedrespectively with histogreen and DAB (magnification 10× and 40× for theinsert box)).

B—Ki67 expression) was analyzed by multi color flow cytometry on Treg(CD4⁺ CD127⁻ CD25^(high)) and Tconv (CD4⁺ CD127⁺ CD25^(low/−)) withinprimary tumor (Ta-Treg, Ta-Tconv) or paired blood (Treg. Tconv).

C—Purified Treg and Tconv from either primary tumor or healthy bloodwere cultured in a 96 well U-bottomed-plate in presence of 500 UI/ml ofIL-2. Cell number was quantified every 4 days by enumeration.

D-F Tumor frozen sections were stained with anti CD3 Ab (brown) andcounterstained with hematoxylin (blue) (10× and 40× in the insert box)(D); CD3 Ab (green) and BDCA2 (brown) (20× and 40× in the insert box)(E); FoxP3 Ab (brown) and BDCA2 (green) (20× and 40× n the insert box)(F).

FIG. 2: ICOS and ICOS-L blockade abrogates IL-10 secretion during pDCmediated T cell activation without interfering strongly on MoDC/Tco-culture. Purified and R848-activated pDC or MoDC were co-cultured for5 days with allogeneic memory CD4⁺ T cells in presence of Ctrl Ab, antiICOS (314.8) or anti ICOS-L (MIH12). At day 5, IL-10 and IFNγ werequantified by ELISA in supernatants from pDC/T co-culture (A) and MoDC/Tco-culture (B).

FIG. 3: ICOS and CD3 co-stimulation favor Treg and Tconv proliferationas well as IL-10 but not IFNγ secretion in the presence of exogenousIL-2.

A/B—FACS-sorted Treg or Tconv issued from tonsil were cultured for 5days alone or with beads coated with CD3/IgG, CD3/88.2, CD3/CD28 agonistmAb in the presence of IL-2 (n=3). The proliferation was assessed by[³11]-Thymidine incorporation (A). IL-10 and IFNγ levels were measuredby ELISA in the culture supernatant (B).

C—CD4⁺ TaT cells sorted from tumor were cultured for 5 days with headscoated with antiCD3/IgG; antiCD3/88.2 or antiCD3/antiCD28 in thepresence of exogenous IL-2 (100 UI/ml). The concentrations of IL-10 andIFNγ in the supernatant were quantified by ELISA.

FIG. 4: ICOS engagement blocks CD28-induced IL-2 and consequentlyreduces proliferation and IFNγ secretion

A—CFSE labeled CD4⁺ memory T cells were cultured for 5 days with thedifferent beads alone or in presence of graded concentration ofexogenous rhIL-2 (20 UI/ml and 100 UI/ml) and proliferation was assessedby CFSE dilution by flow cytometry.

B—IL-2 detected by ELISA after 5 days culture with the different beadswithout exogenous IL-2.

C—Blood CD4⁺ memory T lymphocytes from healthy donors were cultured for5 days with the different heads alone or in presence of exogenous IL-2(100 UI/ml). The IL-10 and IFNγ secretions were quantified by ELISA.

FIG. 5: Absence of expression of ICOS-L on breast tumor cell lines andprimary breast tumor dilacerations

A—ICOS-L expression was assessed by flow cytometry on breast tumorepithelial cell lines suspensions harvested in PBS-EDTA in absence oftrypsin to avoid Ag deterioration.

B—ICOS-L expression was assessed on tumor cells (CD45− cells) after 48 hculture in presence of control Ab (dashed line) or anti ICOS Ab (314.8)(continuous line).

FIG. 6: Treatment of primary Neu15 mammary tumors with a surrogate ratanti-mouse anti ICOS Ab (17G9, IgG2b) slow down the tumor growth.

FIG. 7:

A: Treg cells numbers are increased within primary cervix cancer.

B: Treg cells ICOS+ are increased within primary cervix cancer.

FIG. 8: Increase of ICOS expressing Treg in non Hodgkin lymphoma (NHL)

HD Hodgkin Disease

FL Follicular Lymphoma

DLBCL Diffuse Large B Cell Lymphoma

MCL Mantle Cell Lymphoma

MZL Marginal Zone Lymphoma

FIG. 9: Presence of ICOS+ Treg cells within primary breast tumors has anegative impact on survival

120 paraffin embedded primary tumor samples with 10 years clinicalfollow up were tested for their expression of ICOS using a commercialanti ICOS rabbit polyclonal Ab. Mean of ICOS+ cells were assessed on sixdifferent spots. To perform the statistical analysis the median was usedas cut-off to have equilibrated groups.

Impact of ICOS expression according to the presence of ICOS in theprimary tumor on Overall Survival (A) or Progression Free Survival (B)is shown.

EXAMPLE Example 1: Characterisation of the Antibodies According to theInvention

Material and Methods

I. Cellular Biology

1—Selection/Cell Purification

Isolation of Peripheral Blood Mononuclear Cells

PBMCs (Peripheral Blood Mononuclear Cells) were isolated from peripheralstem cells transplantation of healthy volunteers (Etablissement Francaisdu sang, Marseille, France) by Lymphoprep gradient (Abeys). In tubes:2/3 of blood are deposited dropwise over 1/3 of Lymphoprep andcentrifuged for 20 minutes at 2000 rpm at 20° C. with no acceleration soas not to disturb the gradient. After centrifugation, the mononuclearcells are recovered and washed twice in PBS 1% FCS (Fetal Calf Serum)+heparin for 20 min at 1000 rpm at 20° C.

The cells were then used immediately or frozen at −80° C. to 50·10⁶cells/ml in RPMI 1640 50% FCS 10% DMSO (Dimethyl Sulfoxide). After 24 h,the cells are transferred to the nitrogen for preservation

Negative Selection of CD4

CD4+T lymphocytes were purified from PBMCs. After thawing, the cellswere washed and diluted in 40 μL of sorting buffer (PBS 0.5% BSA 2 mMEDTA) for 10·10⁶ cells. The kit MACS human CD4+ T Cell Isolation Kit IIwere used (Miltenyi Biotec): 10 μL of a solution of monoclonalantibodies conjugated to biotin (primary labeling) are added and themixture was incubated for 10 min at 4° C. with stirring.

The cells are then put in contact with 20 μL of magnetic beads coupledwith anti-biotin (secondary labeling) for 15 min at 4° C. with stirring.After washing with buffer sorting, cells were sorted to Automats(Miltenyi). The negative fraction depleted in CD4+T labeled cells isthen isolated. This gives a population of CD4+ pure of about 95%.

2—Activation and Cell Culture

Pre-Activation with Heads CD3/CD28 and then Stimulation with mAbs

The CD4+ T cells are put to the concentration of 10⁶ cells/mL RPMI 10%FCS in the presence of beads CD3/CD28 (Dynabeads, Invitrogen) (1 cell/1bead) and incubated for 48 h at 37° C. The cells are then separated fromthe heads with a magnet and Dynal Biotech rest overnight in RPMI 10% FCSat a concentration of 10⁶ cells/ml.

On the other hand, mAbs anti-CD3 (OKT3), anti-ICOS (ICOS 88-2), andcontrol IgG1 mAb (Sigma) were coated on a 96 flat well overnight at 4°C. The wells are coated with 50 ng/ml anti-CD3 supplemented with 20μg/ml in other mAbs PBS 1×100 μL/wells. The next day, the plate iswashed with PBS, saturated two hours with PBS 5% FCS (200 μl per well).The CD4+ T cells with the previously incorporated CFSE (see below) aredistributed on the coated plate at a rate of 2105 cells/200 μL ofmedium/well and incubated for 72 h at 37° C. At 48 h, supernatants werecollected and at 72 h. cells were collected to analyze proliferation byflow cytometry (FIG. 4).

Activation by Artificial APC

Magnetic beads (Dynabeads M-450 Epoxy, Invitrogen) were washed in sodiumphosphate buffer 0.1 M and then incubated with mAbs anti-CD3 (OKT3) at aconcentration suboptimal of 1 mg/1·10⁷ heads representing 5% of the mAbscoupled with the beads, with the mAb anti-CD28 or ICOS (ICOS 88-2 orCD28.2), (2 μg/1·10⁷ beads, 10%). These artificial APC were incubatedwith mAbs in slow rotation overnight at 4° C. The next day, two washesare performed in PBS 0.1% BSA. Artificial APC are distributed on a onebead for a cell in a 96 round plate wells on which 2·10⁵ lymphocytes TCD4+/200 μl per well were deposited and then incubated for 72 h at 37°C. The CD4+ T cells have previously incorporated the CFSE. At 48 h,supernatants were collected and 72 h, cells were collected to analyzeproliferation by flow cytometry.

3—Cell Proliferation

The proliferation of lymphocytes is followed by the CFSE(carboxyfluorescein diacetate succinimidyl ester) (Molecular Probes,Invitrogen). The CFSE is cell permeable and nonfluorescent. Whenentering the cell, esterases cleave the acetate groups which becomefluorescent whereas the cell become impermeable.

The characteristic of CFSE is to be shared equitably in each newlyformed cell at each division. It emit in the green radiation allows thesimultaneously analyze of the number, the position and the stage ofdifferentiation of the cells, the fluorescence intensity per cell beingproportional to the concentration of CFSE. To label the cells with CFSE,the cell suspension is diluted in cold 1×PBS. Adding the CFSE: 5 μM to10·10⁶ cells. The cells are then placed in a water-bath at 37° C.

After 8 to 10 minutes stirring, the cells were quickly placed on ice tostop the reaction. The cells are then centrifuged twice with 2 ml of PBS1×. Finally they are collected in the desired volume of RPMI 10% FCS forculture. The proliferation is determined thanks to flow cytometry.

II—Flow Cytometry

CD4+ cells are diluted with 30% BSA PBS (50 μL/well) in a 96-well platefor 10 minutes at 4° C. to saturate nonspecific sites. They are thenincubated for 30 minutes at 4° C. in the dark, with the desiredantibodies coupled to a fluorochrome.

After two washes in PBS 1×1% BSA 0.02% azide (centrifugation 2100 rpm, 3min at 4° C.), cells were fixed in 200 μL of PBS 2% formaldehyde andplaced in a flow cytometer (FACS Canto, BD Biosciences). The results areanalyzed thanks to the FlowJo software.

III—ELISA (Enzyme Linked Immunosorbent Assay)

Culture supernatants of CD4+ T cells are collected at 48 h and stored at−20° C. for an assay on IL-10, IFNγ and TNFα.

Results

1—Characterization of Anti-ICOS mAbs

The inventors developed 5 anti-ICOS Abs. Their isotype was assayed byELISA. For obtaining an indirect analysis of their affinity for theirreceptor, mAbs were tested using stable transfectants expressing ICOS.JICOS.1 cells were in the presence of an increasing range of anti-ICOSmAbs labeled with a probe coupled to a fluorochrome (PE-GAM: Goat antimouse-PE) and the analysis was made thanks to cytometry flow.

It was thus possible to determine the ED 50, i.e. the concentration ofmAbs which 50% of sites are saturated. mAbs with the lowest ED 50 arethose with the highest apparent affinity.

Then the inventors tested the ability of anti-ICOS mAbs to inhibit thebinding of ICOS L (a recombinant form of human IgG1 Fc domain) carriedby the JICOS.1 cell.

They used a gradient of concentration of anti-ICOS mAbs and they reveledthe fixation to ICOS L Fc thanks to a probe coupled to a fluorochrom(GAH-PE: Goat anti-human-PE). The analysis was made by flow cytometry.The inventors thus determined the ID 50 i.e. the dose which inhibits 50%of the binding of ICOS L-Fc on ICOS.

The more the ID 50 is little, the more mAb easily compete withrecombinant ICOS Fc.

The inventors thus evidenced that ICOS R 314-8 and ICOS R 53-3 have ahigh affinity for their binding site (ED 50<0.5 ug/ml) and a significantblocking potential (ID 50<1 mg/ml).

The antibody ICOS R 314-8 was therefore chosen for being coupled to thefluorochrome Alexa Fluor 647 and used in flow cytometry analysis.

2—Anti-ICOS mAbs Differ in their Ability to Induce the Production ofIL-10 by Activated CD4+ T Cell

The inventors tested the ability of the mAbs to act as agonistantibodies, i.e. being able to have the same action as the naturalligand of ICOS, using functional tests. The studied parameter studiedwas the secretion of IL-10 since ICOS induces the production of IL-10 byLT.

The agonistic potential of anti-ICOS mAbs were tested on CD4+ T cells,which were pre-activated with CD3/CD28 beads for 48 h and distributed ona plate where anti-CD3 mAb were coated for continuing the stimulationalong with the various anti-ICOS mAbs.

The culture supernatants were then assayed for 48 h for IL-10 and thesecretion of IL-10 induced by the different anti-ICOS mAbs was comparedbased to the secretion of IL-110 induced by a commercially availableanti-ICOS mAb (ICOS c)

The anti-ICOS mAbs 53-3, 88-2 and 92-17 significantly increased IL-10secretion of CD4+ and thus are agonist antibodies. Regarding, anti-ICOSmAbs 145-1 and 314-8, no significant increase in the production of IL-10was detected.

The inventors finally showed that anti-ICOS mAbs 53-3, 88-2 and 92-17are better agonists than the commercially available anti-ICOS. Indeed,if one considers the commercially available anti-ICOS mAb as reference,the anti-ICOS mAb 88-2 causes increased secretion of IL-10 of +61%,anti-ICOS mAb 92-17 of +20% and anti-ICOS mAb 53-3 of +14%.

The results are summarized in the following table:

[ED] 50 [ID] 50 Agonist mAb Isotype (μg/ml) (μg/ml) effect ICOS 88-2IgG1- L 1.60 17 +++ ICOS 314-8 IgG1- K 0.06 0.29 −

Example 2: Use of an Antagonist Antibody of the Invention and an AgonistAntibody of the Invention

Material and Methods

Immunohistochemistry

Frozen primary breast tumor sections were stained with mouse anti-FOXP3,or anti-Ki67 and revealed using the ImmPRESS anti-mouse Ig peroxidasekit (Abcys, Paris. France) according to the supplier's instructions andDAB. Then, the second primary antibody (mouse anti-ICOS (53.3),anti-CD3, anti-BDCA2 was added and revealed with ImmPRESS kit andHistogreen (Abcys). The specificity of the staining was assessed usingmouse isotype controls in place of the first or the second primaryantibody.

Purification of Mononuclear Cells from Breast Tumors, Tonsils andHealthy Blood

Mononuclear cells (MNC) were purified, from healthy peripheral bloodobtained from EFS or from enzymatic dilaceration of primary breasttumors or tonsils samples, by Ficoll density gradient centrifugation.

Phenotypic Analysis of pDC and T Cells Subsets

For extensive phenotypic analysis, pDC were identified among total MNCas CD4⁺ CD123⁺ cells using FITC or PE anti-CD123 and PE-Cy5 anti-CD4 andPE-coupled antibodies against CD40, CD86 or ICOSL. T cells wereidentified as CD3⁺ CD4⁺ cells. Treg were identified either by the multicolor phenotype CD4⁺ CD127⁻ CD25^(high) or for their FoxP3 expressionafter gating on CD3⁺ CD4⁺ T cells.

Proliferation of Ta-Treg and Ta-Tconv or their blood counterpart wasassessed by multicolor analysis allowing Treg CD4⁺ (CD127⁻ CD25^(high))and Tconv (CD4⁺ CD127⁺ CD25^(Low/−)) characterization associated withKI67 Ab staining.

Flow cytometric analysis was performed on a FACScan (BD Biosciences) oran ADP Cyan (Beckman Coulter) and data were analyzed with Cell Quest Prosoftware (BD Biosciences) or FlowJo (Treestar).

Purification of pDC

pDC were purified from lineage(Lin)-negative enriched MNC by eithermagnetically activated cell sorting using CD304/BDCA-4 microbeads kit ornegative depletion using pDC isolation kit (Miltenyi Biotec)) orFACS®-sorting (FACSVantage SE™ DiVa flow cytometer, BD Biosciences) asLin⁻ CD4⁺ CD11c⁻ cells. Purity was routinely >98%.

In Vitro Generation of Monocytes Derived DC (MoDC)

MoDC were obtained from blood purified monocytes after 7 daysdifferentiation in GM-CSF (100 ng/ml)+IL-4 (50 UI/ml) (Schering Plough,Kenilworth USA).

CD4⁺ Memory T Cells and Treg Purification

Memory CD4⁺ T cells (>95% purity) were obtained from MNC after magneticdepletion including anti-CD45RA Ab, as described (Gobert et al, 2009).CD4⁺ CD25^(high)CD127⁻ Treg and CD4⁺ CD25⁻ CD127^(low/+) conventionalCD4⁺ T cells were FACS®-sorted from purified memory CD4⁺ T cells (Purity>98%).

To follow their in vitro proliferation, purified memory CD4⁺ T cellswere stained at day 0 with CFSE. Viable cells were selected by DAPIexclusion or Live and Dead reagent in case of cell permeabilisation(200,000 and 5,000 minimum events were gated on the total cellpopulation and on purified cells respectively).

DC-T Cell Co-Cultures

Allogeneic memory CD4⁺ T cells, Treg or CD4⁺ Tconv cells were culturedat 3×10⁴ to 5×10⁴ cells in complete medium with IL-2 (100 IU/ml) andhighly purified TApDC, healthy pDC or MoDC that were pre-activated for24 h with IL-3, GM-CSF (10 ng/ml) in the presence of R848. Addition of Tlymphocytes on pre-activated DC subsets was done in triplicate in96-well round-bottomed plates at a ratio of 1:5 (DC/T cells) andco-cultured for 5 days. Proliferation was assessed either by CFSEdilution in experiments analysing FoxP3 expression or by DNA synthesisanalyzed by ³H-TdR uptake.

The impact of ICOS/ICOSL engagement was assessed by addition of ctrl Ab,commercial (ISA-3) or proprietary (314.8) anti ICOS Ab or anti ICOSL(MIH12) in the cultures. To assess T cell cytokines secretion by ELISA,cells were co-cultured with pDC or TApDC, and supernatants harvested atday 5 were centrifuged and stored at −20° C.

Stimulation of Tconv and Treg with Artificial Beads

Artificial APC were produced as described in example 1. Treg (3×10⁴) orTconv (1×10⁵) sorted from tonsil or Ta-CD4⁺ T cells (1×10⁵) purifiedfrom tumors were cultured for 5 days with artificial beads at a ratio1:1 (artificial APC: T cell) in the presence of IL-2 (100 UI/ml) in 96U-bottomed wells under 200 μl. Proliferation was assessed either by CFSEdilution or by DNA synthesis analyzed by ³H-TdR uptake.

Cytokines Detection in T Cell Cultures Supernatants by ELISA

IL-10, IFNγ and IL-2 in 5 days culture supernatants were quantified byELISA using commercial kits from Bender Medsystems according tomanufacturer's instructions.

Result

The data presented below are intended to analyze the impact of twoantibodies against ICOS (i.e. blocking MAb 314.8; agonist MAb 88.2)developed by the inventors in order to validate

i) the blockade of ICOS by antagonistic 314.8 MAb as a new promisingdrug candidate to abrogate the immunosuppressive response observed inbreast cancer; and

ii) the engagement of ICOS by the agonist 88.2 MAb on CD4⁺ T cells tofavour the amplification of Treg that would be of interest in the fieldof autoimmunity.

Ta-Treg that Highly Express ICOS are Present within Lymphoid Aggregatesin Primary Breast Tumors and Proliferate In Situ

The inventors have previously demonstrated the presence of Tumorassociated regulatory T cells (Ta-Treg) expressing CD25^(high) and FoxP3in primary breast tumors within lymphoid aggregates correlating with apoor prognosis and increased risk of metastasis (Gobert et al., 2009).These Ta-Treg represent 15% to 25% of total CD4⁺ TaT cells, are highlyactivated as they express ICOS, CD39, GITR and HLA-DR and suppressTaTconv proliferation and cytokines secretion (IL-2, IFNγ).

These Ta-Treg proliferate within the primary breast tumor environment insitu (Gobert et al., 2009) as demonstrated by either the presence ofICOS⁺ Treg co-expressing Ki67 on tumor frozen sections, (FIG. 1A) or thehigher proportion of KI67⁺ cells within purified Ta-Treg and Treg fromblood (respectively 8% and 4%) compared to Ta-Tconv and Tconv (3% and0.3% respectively) (FIG. 1B).

In contrast with these in vivo results, the inventors demonstrated thatin vitro stimulation of purified Ta-Treg with expand beads (beads coatedwith agonist anti CD3 and anti CD28) is not capable to favor Ta-Tregamplification in contrast to that observed with purified TaTconv orpurified Treg or Tconv from blood of healthy donors (FIG. 1C).

The inventors hypothesized that the ICOS engagement is essential forTa-Treg proliferation and functions.

A) Use of an Antagonist Antibody of the Invention

Blockade of ICOS/ICOS-L Interaction Through Antagonist ICOS mAb (314.8)

Ta-Treg Interact In Situ with Ta pDC within Lymphoid Aggregates inPrimary Breast Carcinoma

Several studies reported the expression of ICOS-L, the specific ligandof ICOS, on pDC (Janke et al., 2006). Using immuno-histochemistry ontumor frozen sections, the inventors observed that Ta-CD3⁺ T cellpresent within the lymphoid aggregates surrounding the tumor are ininteraction with Ta-pDC BDCA2⁺ (FIGS. 1D and 1E). A double staining withFoxP3 and BDCA2 Ab revealed that Ta-Treg are in close contact withTa-pDC in these lymphoid aggregates, suggesting that this interactionwill favor the ICOS engagement by ICOS-L in tumors (FIG. 1F).

Ta-pDC are Activated but Did not Express ICOS-L as a PotentialConsequence of In Situ ICOS/ICOS-L Interaction

After purification from tumor disaggregation, Ta-pDC show an activatedphenotype as they express up regulated levels of CD86 and CD40 comparedto healthy blood and matched patient's blood pDC. As reported by severalgroups (Ito et al., 2007; Janke et al., 2006), freshly isolated healthyblood pDC express low levels of ICOS-L that is strongly unregulatedafter IL-3 exposure or TLR7/8 ligation, which is not observed on otherDC subsets (mDC, MoDC). Interestingly, contrasting with their activatedstatus (CD86⁺ CD40⁺), Ta-pDC are devoid of membrane ICOS-L expression.In contrast freshly isolated paired patients' blood pDC or healthy bloodpDC express ICOS-L. After a 24 h culture period in IL-3 or upon TLR7/8ligation, sorted Ta-pDC reacquire a strong ICOS-L expressiondemonstrating their ability to modulate this ICOS-L expression (data notshown). Among CD3⁺ TaT cells, ICOS is strongly expressed on Ta-Treg(69.9% MFI: 361) in contrast to TaTconv (23% MFI: 83) or TaCD8⁺ (2% MFI:50). These results indicate that in situ ICOS/ICOS-L interaction leadsto ICOS-L down regulation on Ta-pDC membrane.

Blockade of ICOS/ICOS-L Interaction Through Antagonistic Anti ICOS MAb(314.8) Abrogate ICOS-L Down Regulation on pDC

To test this hypothesis, healthy blood T cells were cultured withTLR7-activated pDC purified from tonsil. The inventors observed after 24h culture period with increased T:pDC ratio a dose dependent ICOS-Ldownregulation on pDC. Interestingly the addition of our antagonist MAbagainst ICOS (314.8) abrogates totally this ICOS-L downregulation onpDC, result that is not reproduced using commercial anti ICOS antibody(ISA-3) (data not shown).

The results demonstrate Ta-pDC and Ta-Treg interactions throughICOS/ICOS-L and indicate that ICOS ligation could be involved in Ta-Tregactivation and proliferation.

Coculture of CD4⁺ T Cells as Well as Purified Treg with Activated pDCbut not MoDC Induced Treg Proliferation that is Blocked with 314.8

To test the ability of ICOS/ICOS-L interactions to induce Tregamplification, the inventors cultured total memory CD4⁺ T cell withhealthy blood purified allogeneic TLR7/8 (R848)-activated pDC or mDC.Among purified CD4⁺ T cells, 3.5% expressed FoxP3 (data not shown).After 5 days of co-culture with pDC the proportion of FoxP3^(high)expressing cells, corresponding to Treg, rises to 12.3% and the additionof 314.8 Ab blocks by 80% this enrichment in FoxP3^(high) cells. Incontrast, coculture of CD4⁺ T cell with activated mDC was not able tofavor a distinct FoxP3^(high) subpopulation among CD4⁺ T cells, and theaddition of 314.8 has no significant effect (6.3% to 8%).

Similar results were obtained with CD4⁺ T cells purified from tumor.Ta-Treg Foxp3⁺ represent 9% of freshly purified CD4⁺ TaT cells (data notshown). Their co-culture with R848 activated pDC increases theproportion of Ta-Treg to 14.5% whereas the addition of 314.8 leads to adecrease of Ta-Treg proportion to 4.5%, below the starting level.

FACS sorted purified Treg or Tconv populations stained with CFSE werecultured with R848-activated pDC or LPS-activated MoDC to analyze theirproliferation capacity by flow cytometry (dilution of CFSE expression).First, the inventors observed that in absence of exogenous IL-2, thatactivated MoDC do not induce purified Treg proliferation whereas Tconvstrongly proliferate. In contrast coculture with activated pDC is ableto induce a strong proliferation of both purified Treg and Tconv.

The addition of anti-ICOS 314.8 MAb strongly reduces Treg and Tconvproliferation when pDC are used as APC whereas Tconv proliferation isunchanged in cocultures with MoDC. In this experiment, ICOS or ICOS-Lblockade with commercial antibodies (ISA-3 mAb or MIH-12 MAb) do notaffect neither Treg nor Tconv proliferation in pDC/T co-cultures.

These data demonstrate that the anti-ICOS 314.8 MAb neutralizes ICOSengagement on Treg and abrogates their expansion induced by pDC.

ICOS and ICOS-L Blockade Abrogate IL-10 Secretion During pDC Mediated TCell Activation without Interfering Strongly on MoDC/T Co-Culture.

314.8 MAb also reduces Tconv proliferation in response to activated pDCstimulation. The inventors determined the impact of 314.8 on IFNγ andIL-10 secretion by Elisa during Tconv and allogeneic R848 activated pDC(FIG. 2A) or and LPS activated MoDC (FIG. 2B) co-cultures. In thesesettings IL-10 secretion is totally abrogated by 314.8 mAb (217+/−31pg/ml in control and 13+/−6 pg/ml with 314.8). Whereas, the IFNγsecretion is slightly reduced upon the addition of 314.8 MAb onco-cultures with pDC (32% reduction 507+/−53 pg/ml in control conditionand 341+/−73 pg/ml with 314.8) (FIG. 2A). In Tconv/MoDC co-cultures ICOSinhibition leads to slightly increased secretions of IL-10 and IFNγ(FIG. 2B).

B) Use of an Agonist Antibody According to the Invention

Use of Agonist Anti ICOS MAb (88.2) to Mimick ICOS Engagement

To perfect their understanding on ICOS functions on Treg and Tconv, theinventors generated a model of artificial APC using beads coated withagonist MAbs leading to CD3 (OKT3); CD28 (CD28.2) and/or ICOS (88.2,Table 1) signaling on purified T cells.

ICOS Engagement with an Agonist MAb (88.2) on Treg Induced theirProliferation and their Capacity to Secrete High Amounts of IL-10

First the inventors observed that Treg from healthy donors proliferatein response to anti CD3/88.2 beads in presence of exogenous IL-2 (FIG.3A). As previously reported (Simpson et al., 2010; Ito et al., 2008)upon activation through TCR and ICOS engagement in presence of IL-2,both purified Tconv and Treg subpopulations secrete high amounts ofIL-10 (311+/−22 pg/ml and 426+/−48 pg/ml respectively) and low levels ofIFNγ (205+/−8 pg/ml and 381+/−12 pg/ml). This result contrasts with dataobtained using the anti CD3/anti CD28 beads. In this model Tconv secretelarge amounts of IFNγ (1213+/−72 pg/ml) and low levels of IL-10 (69+/−58pg/ml) whereas Treg secrete IL-10 and low levels of IFNγ (422+/−36 pg/mland 305+/−31 pg/ml respectively) (FIG. 3B).

Similar experiments with T cells purified from tumor demonstrated thatCD4⁺ TaT cells produce similar levels of IL-10 in response to ICOS andCD28 while the levels of IFNγ are weaker in response to ICOS compared toCD28 engagement (FIG. 3C).

ICOS Engagement Blocks CD28-Induced IL-2 and Consequently ReducesProliferation and IFNγ Secretion

Whereas, CD4⁺ memory T cells proliferate in response to anti CD3/antiCD28 stimulation independently of exogenous IL-2, no proliferation isobserved in response to anti CD3/88.2 stimulation (FIG. 4A). Theaddition of hIL-2 rescues this proliferation in a dose dependent manner.Interestingly, the co-engagement of ICOS and CD28 in the absence of IL-2reduces significantly the proliferation of CD4⁺ memory T cells comparedto only CD28 engagement, and this is completely rescued in the presenceof 100 UI/ml IL-2. Interestingly, ICOS ligation through 88.2 mAbabrogates IL-2 secretion detected with anti CD3/anti CD28 stimulation(FIG. 4B). Taken together, this argues in favor of a reduced spontaneousIL-2 secretion when ICOS and CD28 are co-engaged compared to the onlyCD28 engagement suggesting an ICOS inhibitory function on CD28-inducedIL-2 secretion.

Moreover, even in the presence of exogenous IL-2, the inventors observeda 50% reduction of IFNγ produced by Tconv, when ICOS and CD28 aretriggered compared with anti CD3/anti CD28 beads (FIG. 4C).

In contrast, whereas IL-10 secretion is strictly IL-2 dependent whencells are activated under ICOS triggering, as previously described (Ito2008, Paulos 2010), the addition of ICOS signal does not affect theIL-10 secretion induced by anti CD3/anti CD28 (FIG. 4C).

All together these results demonstrate that ICOS ligation reduced theability of anti CD3/anti CD28 to favor Th1 polarisation (through thereduced IFNγ production) but sustains the IL-10 production favoring thedevelopment of an immunosuppressive environment.

ICOS Engagement Through 88.2 MAb Increased the Treg Suppressive Function

To assess that ICOS engagement can be associated with animmunosuppressive T cell response, the inventors setup suppressionsassays in the absence of exogenous IL-2 to compare anti CD3/antiCD28/IgG and anti CD3/anti CD28/88.2 beads efficiency. The addition ofthe ICOS signaling (88.2) strongly increases the suppressive function ofTreg compared with the anti CD3/anti CD28/IgG1 (51% inhibition incondition one Treg for 4 Tconv anti CD3/anti CD28/88.2 compared to 21%inhibition with anti CD3/anti CD28/IgG). All together these resultsdemonstrate that ICOS engagement favors an immunosuppressive T cellresponse that could result either from an increased Tconv sensitivity tosuppression or a stronger Treg suppressive ability.

Example 3: Analysis of Prognostic Impact of Detection of ICOS⁺ TregCells within Primary Breast Tumors

120 paraffin embedded primary tumor samples with 10 years clinicalfollow up were tested for their expression of ICOS using a commercialanti ICOS rabbit polyclonal Ab (Spring Biosciences). ICOS⁺ cells werequantified in double blind on 6 different replicates for each tumor andmean of the results were compiled (data not shown). To perform thestatistical analysis the inventors used the median as cut-off to haveequilibrated groups.

In univariate analysis the inventors demonstrate that the presence ofICOS⁺ cells (>1.66 ICOS⁺ cells/spot) correlated with high tumor grade(p=0.007), expression of Estrogen receptor by tumor cells (p=0.018),luminal A/B molecular subtypes (p<0.001) and absence of Her2/neuoverexpression (p=0.035).

The impact of ICOS⁺ cells detection within primary breast tumors onoverall survival (OS) or progression free survival (PFS) wasinvestigated.

Whereas 6/59 deaths were observed in the ICOS-group, 14/61 patientsdeceased in the ICOS⁺ demonstrating the significant prognostic value ofICOS⁺ detection on OS (Log Rank test p value=0.0465) (FIG. 9A). Similaranalysis performed on PFS demonstrated ICOS⁺ cells was associated with apoorer overall survival, with progression in 11/59 in ICOS⁻ group,whereas 20/61 patients progressed in the ICOS⁺ group (p=0.0285) (FIG.9B).

Example 4: Confirmation of the Existence of In Situ Interaction of pDCwith ICOS⁺ Treg in the Tumor Environment

Ex vivo co-culture of tumor cell dilacerations in the presence of antiICOS 314.8 MAb or Ctrl Ab for 48 h in presence of IL-3 (20 ng/ml). Atthe end of the culture period, the expression of ICOS-L on pDC isobserved only in the presence of the anti ICOS 314.8 MAb and not withcontrol Ab, demonstrating that the down regulation of ICOS-L on pDC ismediated through an interaction with ICOS⁺ cells (data not shown).

Example 5: Epithelial Breast Tumor Cells from Either Established CellLines or Fresh Tumor Samples do not Express ICOS-L in Contrast toMelanoma or Glioma Tumor Cells Even after Ex Vivo Culture with Anti ICOSAntibody 314.8

Breast epithelial tumor cells lines were harvested in PBS EDTA to avoidtrypsin-associated degradation of the Ag and cells were stained withanti ICOS-L antibody to evaluate the expression at cell surface by flowcytometry. None of the cell lines tested was found positive for ICOS-L(FIG. 5A). Similar analyses were performed on 48 h cultured primarytumor disagregations in presence of anti ICOS Ab (314.8) or control Abplus IL-3 (20 ng/ml) (FIG. 5B).

Example 6: Impact of a Surrogate Rat Anti-Mouse Anti ICOS Ab (17G9,IgG2b) on Mammary Tumor Growth in a Syngenic Mammary Tumor Model

Mouse mammary tumor model was obtained in female FVB mice in 28-35 daysafter orthotopic injection of the Neu 15 cell line. The generated tumorsappear significantly infiltrated by activated Ta-pDC, ICOS^(high) TATregand resting TATconv.

Injection of 17G9 antibody (50 μg/ml) intra-peritoneally three times aweek from day 11 after tumor implantation results in a reduced Neu15tumor size at late time points compared to the injection of control Ab(LTF2, IgG2b) (p=0.053) (FIG. 6).

Example 7: A: Treg Cells Numbers are Increased within Primary CervixCancer

Cervical samples were obtained from patients either with dysplasia(CIN2/3, n=18) or cancer (n=14). Normal cervical tissue was used ascontrol (n=11). Samples were obtained by both enzymatic and physicaldissociations. After washing, mononuclear cells were incubated withlabeled mAbs and Tregs enumerated as CD127^(low)/CD25^(bright)CD4⁺ Tcells. The percentage of Treg within the CD4⁺ subset is depicted. Tregwere increased within cervical cancer samples in comparison to normaltissue and dysplasia. Hence, this increase is associated to the cancerdevelopment (FIG. 7A).

B: Treg Cells ICOS⁺ are Increased within Primary Cervix Cancer.

Cervical samples were obtained from patients either dysplasia (CIN2/3,n=5) or cancer (n=12). Normal cervical tissue was used as control (n=5).Samples were obtained by both enzymatic and physical dissociation. Afterwashing mononuclear cells were incubated with labeled using ICOS mAbsand Tregs enumerated. The percentage of Treg ICOS within the CD4⁺ subsetis depicted ICOS⁺ Treg are present within tissues with only a trend totheir increase in cervical cancer due the limited numbers of samplesanalyzed (FIG. 7B).

Example 8: Increase of ICOS Expressing Treg in Non Hodgkin Lymphoma(NHL)

The inventors have analyzed the Teg numbers and the expression of ICOSon Treg in LNH samples. Fresh lymphoma cells teased from lymph nodeswere collected from 45 patients with informed consent. Lymphoma samplescorrespond to Hodgkin disease (HD, n=11), follicular lymphoma (FL,n=13), diffuse large B cell lymphoma (DLBCL, n=10), mantle cell lymphoma(MCL, n=5) and marginal zone lymphoma (MZL, n=6). Detection of Tregcells was performed by incubation for 20 min at 4° C. with anti-ICOS-PE(Becton Dickinson™), anti-CD3-ECD, anti-CD4-Pacific Blue (BeckmanCoulter®), anti-CD127 FITC, anti-CD25 APC-Cy7 and LIVE/DEAD® FixableDead Cell Stain Kit (Invitrogen™). After staining, each cell preparationwas washed twice in PBS, fixed with 2% paraformaldehyde and analyzed ona FACS LSR2 flow cytometer (Becton Dickinson™). Data were analyzed usingFlowJo Software (TreeStar™). Treg were increased in all lymphoma samplesexcept HD. Most of Treg displayed an increased expression of ICOS incomparison to control lymph nodes (FIG. 8).

Example 9: Sequencing of Icos 314.8 (CNCM I-4180)

Total RNA was extracted from provided frozen hybridoma cells and cDNAwas synthesized. Then, RT-PCR was performed to amplify the variableregions (heavy and light chains) of the MAb. The MAb variable regions ofthe heavy and light chains were cloned into a cloning vector separately,then the obtained sequences were analyzed to deduce the sequences of theMAb.

Materials

Hybridoma cells ICOS 314.8 (CNCM I-4180); TRIzol® Plus RNA PurificationSystem (Invitrogen, Cat. No: 15596-026); SuperScript™ III First-StrandSynthesis System (Invitrogen, Cat. No: 18080-051).

Methods

Total RNA Extraction

Total RNA was isolated from the hybridoma cells according to thetechnical manual of TRIzol® Plus RNA Purification System. The total RNAwas checked by gel electrophoresis.

RT-PCR

Total RNA was reverse transcribed into cDNA using isotype specificanti-sense primer or universal primer and whole procedure was accordingto the technical manual of SuperScript™ ITT First-Strand SynthesisSystem. The antibody fragment will be amplified according to thestandard operation protocol of RACE method of GenScript.

Cloning of Antibody Genes

Target PCR products of antibody genes were cloned into the cloningvector separately according to standard molecular cloning procedures.

Screening and Sequencing

Colony screening was employed to screen clones with inserts of correctsizes, and no less than ten independent positive colonies were sequencedfor each antibody fragment.

Results and Analysis

Total RNA Extraction

Total RNA of the sample was run alongside of DL3000 DNA marker on a 1.5%agarose/GelRed™ gel electrophoresis.

PCR Product of Antibody Genes

4 μl PCR products of each sample were run alongside of DL3000 DNA markeron a 1.5% agarose/GelRed™ gel electrophoresis.

Sequencing Results and Analysis

The sequencing results are as follows. The consensus DNA sequences andcorresponding amino acid sequences are listed below:

Heavy chain: DNA sequence (426 bp): Leadersequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 13)ATGGGATGGCGCTGTATCATCCTCTTCTTGGTATCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTACAGCACCCTCCGACTGAACTTATGAACCCTCGCCCTTCACTGAACCTGTCCTGCAACCCTTCTGGCTACACCTTCACCACCTACTGGATGCACTGGGTGAACCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATCCTTCTGATAGTTATGTTAACTACAATCAAAACTTTAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATACAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTTTGTGCGAGATCCCCTGATTACTACGGTACTAGTCTTGCCTGGTTTGATTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTACAHeavy chain: Amino acids sequence (142 AA): Leadersequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 14)MGWRCIILFLVSTATGVHSQVQLQQPGTELMKPGASVKLSCKASGYTFTTYWMHWVKQRPGQGLEWIGEIDPSDSYVNYNQNFKGKATLTVDKSSSTAYIQLSSLTSEDSAVYFCARSPDYYGTSLAWFDYWCQCTLVTVSTLight chain: DNA sequence (396 bp): Leadersequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 15)ATGAGGTGCCTAGCTGAGTTCCTGGGGCTGCTTGTGCTCTGGATCCCTGGAGTCATTGGGCATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCCCCTGCATAGTAACGGCAACATTTACTTATATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTACTTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAALight chain: Amino acids sequence (132 AA): Leadersequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 16)MRCLAEFLGLLVLWIPGVIGDIVMTQAAPSVPVTPGESVSISCRSSKSPLHSNGNIYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTTFTLKISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK

Thus, sequences of ICOS 314.8 (CNCM I-4180) can be resumed as follows:

Aminoacid DNA sequence sequence H-CDR1 GGCTACACCTTCACCACCTACTGGATGGYTFTTYWMH CAC (SEQ ID NO: 7) (SEQ ID NO: 1) H-CDR2GAGATTGATCCTTCTGATAGTTATCTT EIDPSDSYVNYNQN AACTACAATCAAAACTTTAAGGGC FKG(SEQ ID NO: 2) (SEQ ID NO: 8) H-CDR3 TTTGATTAC FDY (SEQ ID NO: 3)(SEQ ID NO: 9) L-CDR1 AGGTCTAGTAAGAGTCCCCTCCATAGT RSSKSPLHSNGNIYAACGGCAACATTTACTTATAT LY (SEQ ID NO: 4) (SEQ ID NO: 10) L-CDR2CGGATGTCCAACCTTGCCTCA RMSNLAS (SEQ ID NO: 5) (SEQ ID NO: 11) L-CDR3ATGCAACATCTAGAATATCCGTACACG MQHLEYPYT (SEQ ID NO: 6) (SEQ ID NO: 12)

Example 10: Sequencing of Icos 88.2 (CNCM I-4177)

Total RNA was extracted from provided frozen hybridoma cells and cDNAwas synthesized. Then, RT-PCR was performed to amplify the variableregions (heavy and light chains) of the MAb. The MAb variable regions ofthe heavy and light chains were cloned into a cloning vector separately,then the obtained sequences were analyzed to deduce the sequences of theMAb.

Materials

Hybridoma cells ICOS 88.2 (CNCM I-4177); TRIzol® Plus RNA PurificationSystem (Invitrogen, Cat. No: 15596-026); SuperScript™ III First-StrandSynthesis System (Invitrogen, Cat. No: 18080-051).

Methods

Total RNA Extraction

Total RNA was isolated from the hybridoma cells according to thetechnical manual of TRIzol® Plus RNA Purification System. The total RNAwas checked by gel electrophoresis.

RT-PCR

Total RNA was reverse transcribed into cDNA using isotype specificanti-sense primer or universal primer and whole procedure was accordingto the technical manual of SuperScript™ III First-Strand SynthesisSystem. The antibody fragment will be amplified according to thestandard operation protocol of RACE method of GenScript.

Cloning of Antibody Genes

Target PCR products of antibody genes were cloned into the cloningvector separately according to standard molecular cloning procedures.

Screening and Sequencing

Colony screening was employed to screen clones with inserts of correctsizes, and no less than ten independent positive colonies were sequencedfor each antibody fragment.

Results and Analysis

Total RNA Extraction

Total RNA of the sample was run alongside of DL3000 DNA marker on a 1.5%agarose/GelRed™ gel electrophoresis.

PCR Product of Antibody Genes

4 μl PCR products of each sample were run alongside of DL3000 DNA markeron a 1.5% agarose/GelRed™ gel electrophoresis.

Sequencing Results and Analysis

The sequencing results are as follows. The consensus DNA sequences andcorresponding amino acid sequences are listed below:

Heavy chain: DNA sequence (429 bp): Leadersequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 29)ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACAGTTTCACCAGCTACTGGATAAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAAATATTTATCCTTCTGATAGTTATACTAACTACAATCAAATGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAACACAGCCTACATGCAGCTCACCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGAATCTTTCTTATTACTTCGATAATAACTACTACTTGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAHeavy chain: Amino acids sequence (143 AA): Leadersequence-FR 1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 30)MGWSCIILFLVATATGVHSQVQLQQPGAELVRPGASVKLSCKASGYSFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQMFKDKATLTVDKSSNTAYMQLTSPTSEDSAVYYCTRWNLSYYFDNNYYLDYWGQGTTLTVSSLight chain: DNA sequence (396 bp): Leadersequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 31)ATGAGGTGCCTAGCTGAGTTCCTGGGGCTGCTTGTGCTCTGGATCCCTGGAGCCATTGGGGATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAACTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGGAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAALight chain: Amino acids sequence (132 AA): Leadersequence-FR 1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 32)MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPWTFGGGTKLEIK

Thus, sequences of ICOS 88.2 (CNCM I-4177) can be resumed as follows:

Aminoacid DNA sequence sequence H-CDR1 GGCTACAGTTTCACCAGCTACTGGATAAAGYSFTSYWIN C (SEQ ID NO: 17) (SEQ ID NO: 23) H-CDR2AATATTTATCCTTCTGATAGTTATACTAA NIYPSDSYTNYNQM CTACAATCAAATGTTCAAGGAC FKD(SEQ ID NO: 18) (SEQ ID NO: 24) H-CDR3 TGGAATCTTTCTTATTACTTCGATAATAAWNLSYYFDNNYYLDY CTACTACTTGGACTAC (SEQ ID NO: 25) (SEQ ID NO: 19) L-CDR1AGGTCTAGTAAGAGTCTCCTGCATAGTAA RSSKSLLHSNGNTYL TGGCAACACTTACTTGTAT Y(SEQ ID NO: 20) (SEQ ID NO: 26) L-CDR2 CGGATGTCCAACCTTGCCTCA RMSNLAS(SEQ ID NO: 21) (SEQ ID NO: 27) L-CDR3 ATGCAACATCTAGAATATCCGTGGACGMQHLEYPWT (SEQ ID NO: 22) (SEQ ID NO: 28)

The invention claimed is:
 1. A method of treatment of cancer in apatient in need thereof, the method comprising administering to thepatient an antibody directed against human Inducible T-cell costimulator(ICOS), wherein said cancer is determined to have regulatory T cells(Treg cells) present in a tumor microenvironment and wherein saidantibody reduces the number and/or activity of the Treg cells in thetumor microenvironment, and wherein said cancer is selected from thegroup consisting of ovarian cancer, cervical cancer, breast cancer,colon cancer, lung cancer, prostate cancer, head and neck cancer,pancreatic cancer, bladder cancer, colorectal cancer, kidney cancer,stomach cancer, skin cancer, and esophageal cancer.
 2. The method ofclaim 1, wherein said antibody reduces the number of Treg cells in atumor microenvironment.
 3. The method of claim 1, wherein said antibodyreduces the activity of Treg cells in a tumor microenvironment.
 4. Themethod of claim 1, wherein said cancer is ovarian cancer.
 5. The methodof claim 1, wherein said cancer is cervical cancer.
 6. The method ofclaim 1, wherein said cancer is breast cancer.
 7. The method of claim 1,wherein said cancer is colon cancer.
 8. The method of claim 1, whereinsaid cancer is lung cancer.
 9. The method of claim 1, wherein saidcancer is prostate cancer.
 10. The method of claim 1, wherein saidcancer is head and neck cancer.
 11. The method of claim 1, wherein saidcancer is pancreatic cancer.
 12. The method of claim 1, wherein saidcancer is bladder cancer.
 13. The method of claim 1, wherein said canceris colorectal cancer.
 14. The method of claim 1, wherein said cancer iskidney cancer.
 15. The method of claim 1, wherein said cancer is stomachcancer.
 16. The method of claim 1, wherein said cancer is skin cancer.17. The method of claim 1, wherein said cancer is esophageal cancer.